**2. Origin and precursors of TCA in cork material and wine**

In order to work on preventive measures against TCA defects, it is important to identify its origin in cork and wine. The presence of hyperhalogenated molecules such as TCA in nature is associated with anthropogenic activities. Precursors of haloanisoles (including TCA) are halophenols (PCP—pentachlorophenol, TCP—2,4,6-trichlorophenol, etc), which for many decades in the twentieth century were widely used as components of chlorophenol-based biocides: herbicides, insecticides, fungicides. These products were extensively utilized in agriculture, for the treatment of wooden materials, cardboard, textiles, etc. [22, 23]. Since that time, the problem of *cork tainted* wines began to attract more and more attention, as mentioned at the beginning of the review. PCP, TCP, and other chlorophenols are relatively stable molecules, but hyperchlorinated phenols can slowly degrade, losing chlorine atoms in the structure (e.g., PCP → TCP). As a result, these compounds can spread and persist in ecosystems for decades and accumulate in cork trees or soil, serving as one of the possible precursors of TCA in cork (**Figure 1)** [22, 23]. Once TCP is in the bark or wood, the formation of TCA occurs microbiologically, which involves *O*-methylation of TCP (**Figure 2**). *Penicillium*, *Fusarium,* and *Trichoderma* strains are considered as microorganisms, which are able to carry out this bioconversion at high and moderate levels [15, 24]. The physiological reason for biomethylation by these filamentous fungi is a defensive response to TCP, which acts as a strong toxin (fungicide). Filamentous fungi are widely spread in nature and do not produce TCA without its precursor TCP. Therefore, the objective reason for the formation of TCA in cork and wood is not the presence of filamentous fungi, but the contamination of these materials with cholorphenols, in particular TCP.

**Figure 2.**

*Microbiological formation of TCA by O-methylation of TCP.*

Besides the fact that TCP and other chlorophenols are banned as biocides in many countries, these compounds can still be found in many places in nature. The latter also include remote areas that have not been directly treated with these biocides, but contaminated by waterways and atmospheric precipitations (**Figure 2**) [25]. In general, a limited number of organisms are capable of transforming halophenols, which results in a low degradation rate of these compounds in nature. In addition, there are also other pathways of TCP accumulation in cork and wooden materials, which are described in the following subsections.

### **2.1 Origins of TCA in cork stoppers**

Exogenous contamination of trees by biocides represents the important origin of TCP in bark and wood. As discussed above, TCP is microbiologically transformed into TCA, the latter accumulates in bark, from which cork stoppers are then produced. However, there are also other sources of TCP and TCA in the cork material, some of which were quite relevant in the past. One of these pathways of TCA formation starts from the *chlorination of phenol* present in cork*.* Phenol is formed in cork and wooden materials by degradation of lignin and by the action of *Penicillium* spp. These fungi are able to synthesize phenol starting from glucose following the pentosephosphate and shikimic acid pathways [26]. Then the treatment of cork with chlorine-containing agents can lead to the chlorination of phenol yielding various chlorophenols, including TCP and dichlorophenols (**Figure 3**). Such cork treatment was widespread before 1990 during the production process of corks:


Chlorination of phenol is a chemical process, however, some authors suggested that biochemical transformation by *Basidiomycetes* can also take place under certain conditions [23]. As was already discussed, the formation of TCA involves the *O*-methylation step, which can occur before or after chlorination of phenol (**Figure 3**). One of the signs of the use of chlorine-containing substances in the manufacture of corks is the presence of other compounds, such as chlorocresols and chloromethylanisoles, which have a moldy off-odor similar to TCA.

Nowadays, in order to protect the quality of cork stoppers, the application of chlorine-based treatments is strongly discouraged by the "International Code of Cork Stopper Manufacturing Practices" promoted by the European Confederation of Cork (C.E. Liège) [7]. The practice of hypochlorite usage as bleaching agent was banned around 1990 and completely abandoned by all cork stopper producers.

*State-of-the-Art Knowledge about 2,4,6-Trichloroanisole (TCA) and Strategies to Avoid… DOI: http://dx.doi.org/10.5772/intechopen.103709*

#### **Figure 3.**

*TCA formation* via *chlorination of phenol in cork and wooden materials (based on [26, 27]).*

Hypochlorite was substituted by hydrogen peroxide H2O2 that does not cause haloanisole problems. The application of chlorine-containing tap water for the bark slabs boiling process is also forbidden. As a result of these measures, along with the improved analytical control, the average cork contamination was significantly reduced, but the TCA problem was not completely resolved.

The other potential source of TCP in cork material is *degradation of PCP*. Among chloroanisole-based biocides, PCP was probably the most utilized. Thus, in the 1970s in the United States alone, its production reached about 23 k tons per year [28]. Unsurprisingly, PCP is still abundant in nature and in wooden materials. In the presence of some bacteria, the reductive dechlorination of PCP occurs as a part of the chlorophenol degradation process (**Figure 4**), which implies replacement of chlorine atoms by hydrogen and formation of less chlorinated phenols.

**Figure 4.** *Dechlorination reactions of PCP and formation of chloroanisoles.*


## **Table 2.**

*Sensory thresholds of haloanisoles in alcoholic solutions (wine).*

Among others, TCP and 2,3,4,6-tetrachlorophenol (TeCP) can be observed as products of dehalogenation [25, 29, 30]. All these chlorophenols can be microbiologically converted to corresponding chloroanisoles: TCA, TeCA, and PCA. The concentration of the latter in wine can be even higher than TCA, however, PCA does not play a prominent role in *cork taint*, since its sensory threshold is higher by 3–4 orders of magnitude and is measured in μg/L (**Table 2**).

Given the different origins of TCA in cork stoppers, it is sometimes unclear which pathway contributes to the formation of TCA in each specific case. The cork stoppers production process (**Figure 5**) includes steps, which are aimed at reducing the TCA content originating from contaminated trees. Among these processes are the aeration of bark slabs, extraction of contaminants by boiling of bark slabs in water, etc. However, all these efforts to reduce TCA may be futile if the succeeding production steps are poorly controlled. For example, TCA can be subsequently regenerated in the treated cork material if the bark slabs are stored and transported wet. Under these conditions, fungi develop rapidly and biomethylation of TCP leads to reappearance of TCA. Therefore, it is necessary to strictly monitor all critical stages in the cork stopper production. Over the past decades, many efforts and technological improvements have been implemented by cork producers to reduce

**Figure 5.** *Typical steps in the production of natural cork stoppers (\*more details in section 4).*

*State-of-the-Art Knowledge about 2,4,6-Trichloroanisole (TCA) and Strategies to Avoid… DOI: http://dx.doi.org/10.5772/intechopen.103709*

and control the fungi growth, prevent the TCA formation in cork material and its removal during the production process.

Finally, if contaminated corks are detected, the origin of TCA can be deduced from the simultaneous analysis of haloanisoles, halophenols, and their ratio. For example, the presence of dichlorophenols in cork or tainted wine indicates the probable involvement of chlorine at some stages of cork stopper production (**Figure 3**) rather than TCP precursor from the forest [23].

#### **2.2 Other sources of TCA in wine**

Musty/moldy defects in wine caused by TCA cannot be attributed only to cork stoppers. There are cases when wines are bottled with plastic closures or screw caps and can still be contaminated with TCA. These incidents have happened in the past and continue to surprise wine producers and consumers today. Possible ways of such contamination are as follows:

• *Contaminated air and winery equipment*. Formation of haloanisoles, including TCA, is possible directly in wine cellars. Corresponding precursors, TCP and other chlorophenols, can be present in various wooden elements: roof constructions, walls, floor, paints, pallets, barrels, etc. [32, 33]. These precursors often originate from chlorophenol-based biocides, which were used in the past as fungicides for wood protection or paint preservatives, or are formed from the reactions of chlorine-containing detergents with wood components in the cellar, as shown in **Figure 3**. Then, filamentous fungi produce TCA (**Figure 2**), which is volatile and contaminates the air. Subsequently, TCA can be easily absorbed by winery equipment, plastic hoses, filter sheets, bentonite, wooden barrels, various enological products, and transmitted to the wine once it gets in contact with the contaminated surfaces (**Figure 6**). The described scheme of wine contamination is more typical for old cellars, where wooden constructions, paints, plasters, walls can contain remarkable quantities of chloroanisole precursors. Nowadays, these compounds are forbidden as biocides, however, other risks of air contamination also exist in modern cellars. Bromophenol-based biocides (2,4,6-tribromophenol, TBP) are still allowed for the wood treatment and can be present in paints, resin laminates, etc. [34]. Similar to the reaction in **Figure 2**, filamentous fungi are able to convert TBP to 2,4,6-tribromoanisole (TBA), which has analogous sensory properties as TCA: musty/moldy off-odor and low sensory perception threshold (**Table 2**). Therefore, the current analysis of musty/ moldy wines usually includes the determination of not only TCA and chloroanisoles, but also TBA. Once the source of TCA or TBA in the cellar is identified, it should be eliminated. If it is not possible and the air contamination is not very high, then intensive air ventilation may be the solution. Among the preventive measures is the replacement of wooden elements in the cellar, e.g., metallic or plastic pallets instead of wooden ones. The utilization of chlorinecontaining detergents to clean the winery and equipment should be avoided. Finally, it is recommended to periodically check the air in the cellar for various contaminants. The standardized method of halophenols and haloanisoles analysis in air involves passive sampling by bentonite spread out over a strip of aluminum foil and exposed to the atmosphere for at least 5 days [35]. Then the contaminants are extracted by ether/hexane mixture (or other solvents) and analyzed by GC–MS. Active sampling methods were also suggested, e.g., pumping air through the tubes with Tenax TA™ sorbent followed by thermal desorption – GC – triple quadrupole MS [36].

**Figure 6.** *Possible ways of wine contamination with TCA in a cellar.*


into wine. Several studies demonstrated that different grades of natural and agglomerated corks are excellent barriers against airborne *d*5-TCA for at least 2–3 years of bottle storage in a contaminated atmosphere [40–43]. The analysis of these stoppers revealed that *d*5-TCA was detected only on the top of the closures, which was in contact with the contaminated air. As for other types of closures, certain amounts of airborne *d*5-TCA were found in wines sealed with some types of synthetic stoppers, glass stoppers, and screw caps (excluding those with Tin Saran liner). One of the possibilities to protect wines with plastic stoppers from the airborne haloanisoles contamination is to use capsules without holes. This approach allowed to reduce the wine contamination with airborne *d*5-TCA by about 10 times or more [44]. A possible criticism of many of these studies about the migration of TCA through bottle closures is that the applied storage conditions involved relatively high levels of air pollution. At the same time, there are no comprehensive reviews summarizing the TCA levels in air in real polluted environments. As for real cases of wine contamination *via* this mechanism, one of them was described in the Annual Report of Australian Wine Research Institute [45]. A large batch of sparkling wine with crown seals (about 14 months after *tirage*) was analyzed because of the musty taint, and the presence of TeCA and traces of PCA was determined. As a result of the investigation, it was suggested that several months of exposure to the contaminated air allowed the migration of TeCA through the crown seals in quantities sufficient to taint the wine. Wood preservatives were identified as a potential source of haloanisoles.

Given all of these potential pathways for TCA contamination, there is a need to more comprehensively investigate the problems associated with musty/moldy wines rather than simply linking them to cork stoppers.
